JP5153068B2 - Analyzing biological interactions - Google Patents

Description

  The present invention relates to a method for analyzing biological interactions related to various living bodies. The present invention relates to an analysis method applicable to, for example, a reporter assay for quantitative detection using bioluminescence regarding intracellular interactions.

In recent years, in research fields such as cell biology and molecular biology, GFP (Green Fluorescent Protein) and luciferase gene, which is a bioluminescent enzyme, act as an expression reporter to fluoresce at specific sites and functional proteins in cells. There is an increasing need to observe biological cells with labels and luminescent labels.
In the observation using GFP, GFP is a protein that emits fluorescence in response to irradiation of excitation light, and the sample that has acted on GFP is irradiated with high intensity excitation light to obtain fluorescence. Observation for about 1 to 2 hours is the limit, but in observation using luciferase, luciferase is a self-luminous enzyme and does not require excitation light that damages the specimen, so observation for about 5 days is possible. In addition, since the fading of fluorescence proceeds by a plurality of excitation light, there is one aspect that is not suitable for long-term or continuous analysis. Furthermore, since variations in fluorescence intensity are likely to occur, even if an improvement that corrects or avoids the influence of fading can be realized, quantitative evaluation depending on the fluorescence intensity is virtually impossible.

  When the luciferase gene is introduced into a cell as a reporter gene and the intensity of expression is examined using luciferase activity as an indicator, the effect on the transcription of the DNA fragment can be determined by connecting the target DNA fragment upstream or downstream of the luciferase gene. You can investigate. In addition, by co-expressing a gene such as a transcription factor that is thought to affect transcription and connecting it to an expression vector, the effect of the gene product on the expression of the reporter gene can be examined. Furthermore, unlike fluorescence, light emission due to a luminescence reaction has almost no variation, which is suitable for quantitative evaluation. Methods for introducing a reporter gene such as a luciferase gene into cells include the calcium phosphate method, the lipofectin method, and the electroporation method, which are properly used depending on the purpose and cell type. As a technique for quantitatively monitoring the luciferase activity introduced and expressed in cells, a lysate containing a large number of cells is reacted with a substrate solution containing luciferin, ATP, magnesium, etc., and a photomultiplier tube is used. The amount of luminescence is measured by the used emission spectrometer (hereinafter referred to as a luminometer) (Patent Document 1). Since a large number of cells (6 million cells) are allowed to luminescence in the wells, the cells are lysed, and the cell solution is replaced with a measurement tube to measure the amount of light in the luminometer. Is measured as the average value of the whole cell.

JP-A-2005-192565

  By the way, when the luciferase gene is introduced into a cell as a reporter gene and the intensity of luciferase gene expression is examined using the amount of luminescence from the cell as an index, the target DNA fragment is connected upstream or downstream of the luciferase gene. Thus, the influence of this DNA fragment on the transcription of the luciferase gene can be examined. In addition, by connecting a gene such as a transcription factor that is thought to affect transcription of the luciferase gene to an expression vector and co-expressing it with the luciferase gene, the influence of the gene product on the expression of the luciferase gene can be examined. Methods for introducing a reporter gene such as a luciferase gene into a cell include the calcium phosphate method, the lipofectin method, and the electroporation method. These methods can be used depending on the purpose of introduction and the type of cell. In the measurement of the amount of luminescence from cells by luciferase activity, the amount of luminescence is quantified by a luminometer using a photomultiplier tube after reacting the cell lysate with a substrate solution containing luciferin, ATP, magnesium, etc. . In this measurement, since the amount of luminescence is measured after the cells are lysed, the expression level at a certain point in time is measured as an average value of the whole cell.

  In addition, in order to capture changes in gene expression over time, it is necessary to measure the amount of light emitted from living cells in time series. For example, measuring the expression rhythm with a certain periodicity by providing a luminometer function in an incubator for culturing cells and measuring the amount of light emitted from the whole cell population being cultured at regular intervals. Can do. In this case, the change in the amount of expression over time due to the total amount of luminescence generated from many cells and other components is measured.

  On the other hand, when the gene expression is transient, the expression level in individual cells varies greatly. For example, even in the case of cloned cultured cells such as HeLa cells, the response of drugs via receptors on the cell membrane surface may vary among individual cells. That is, even if a response as a whole cell is not detected, several cells may be responding. In this case, it is important to measure the expression level in individual cells, not from the whole cells.

  Furthermore, since the luminometer can use a cell solution containing a large number of cells only at one measurement time point, a minimum number of cells and a container corresponding to the number of times corresponding to the time to be measured are required. Moreover, the same cells cannot be continuously evaluated by preparing separate cell solutions for different measurement time points.

  Therefore, an object of the present invention is to detect an arbitrary interaction in a biological sample directly and minimally invasively. Another object of the present invention is to provide a method for analyzing interactions inside and outside cells or tissues with high accuracy by luminescence analysis, which is stable quantitative analysis that is particularly difficult with fluorescence analysis. Another object of the present invention is to provide a method for selecting only luminescent cells and accurately analyzing the amount of luminescence, position, morphological information, etc. for cells that have been reliably transfected.

In order to solve the above problems, the analysis method of the present invention is a method for analyzing an interaction between cells and tissues, and a luminescent substance as a biological excitation substance for at least one of the interactable substances Using an objective lens having a value of (NA / β) ² of 0.01 or more when the interacting substance labeled with is present in a plurality of cells and the numerical aperture and the projection magnification are expressed as NA and β, respectively. and characterized in that it comprises a while optically form an image by an image pickup field of view included the emission as a result of the interaction plurality of cells captured and analyzed separately a plurality of cell images in the same field of view captured step To do. Here, the presence and / or amount of weak light in the individual cell image is preferably correlated with the interaction. It is also preferred that the biological excitation material includes a component that provides bioluminescence. More preferably, the component that provides bioluminescence uniformly contains a substrate for the luminescent reaction.

    In addition, the method of the present invention preferably includes a step of evaluating an interaction with a chemical stimulant that affects the activity of the biologically excited substance. Preferably, the method further includes a step of evaluating an interaction with a physical stimulus that affects the activity of the biologically excited substance. Further, it is more preferable that the biological sample contains living cells. Moreover, it is preferable that the said analysis process includes the measurement process which measures several cells in the same visual field statistically. Here, it is more preferable that the measurement step includes a step of calculating at least the number of cells that generate detectable luminescence. Further, it is more preferable that the counting step includes a step of sorting by group according to the amount of luminescence for each cell. In any case, it is preferable to include a step of expressing the result information by the measurement step in association with the cell image so as to be visible. Instead of the expressing step, the imaging step is executed at a number of different timings, and the result information from the measuring step is output in real time along the time axis or synchronized along the activity curve of the luminescence reaction. This is also preferable when it includes a step of outputting the output.

Further, the analysis method of the present invention is a method for analyzing an interaction between cells and tissues, wherein an interaction substance obtained by labeling a luminescent substance as a biological excitation substance on at least one of the interactable substances is provided. Use an objective lens that has a value of (NA / β) ² of 0.01 or more when the same cell or multiple parts of the same tissue are present at the same time, and the numerical aperture and projection magnification are expressed as NA and β, respectively. The resulting light emission from the interaction is imaged while optically imaged by the imaging field including the cells or tissues, and the information about the plurality of parts in the same cell or the same tissue is imaged. And a step of analyzing based on the above.

Further, the analysis method of the present invention is a method for analyzing an interaction between cells and tissues, wherein an interaction substance obtained by labeling a luminescent substance as a biological excitation substance on at least one of the interactable substances is provided. Use an objective lens that has a value of (NA / β) ² of 0.01 or more when it exists in a specific location or distribution state of the same cell or tissue, and the numerical aperture and projection magnification are expressed as NA and β, respectively. Then, light emission as a result of the interaction was imaged in time series while optically imaging the imaging field including the cell or tissue, and information on the same cell or multiple sites in the same tissue was imaged. The method includes a step of analyzing a change in a location or a distribution state of the luminescent material based on image information.

Further, the analysis method of the present invention is a method for analyzing interactions inside and outside a cell or tissue, wherein a plurality of types of interacting substances that can interact are labeled with a luminescent substance as a biological excitation substance or the cells or When the numerical aperture and the projection magnification are expressed as NA and β, respectively , the objective lens having a value of (NA / β) ² of 0.01 or more is used to emit light as a result of the interaction. The method includes a step of imaging while optically forming an image in an imaging field including the cells or tissues, and analyzing each of the interacting substances based on the captured image information. Here, it is preferable that the plurality of types of interacting substances are separately labeled with luminescent substances having different optical characteristics that are optically distinguishable. In addition, it is preferable to perform chemical or physical stimulation specific to each of the plurality of types of interacting substances at different timings.

  According to the present invention, it is important in practical use to capture a luminescence image of a cell into which a luciferase gene has been introduced with an optical system that satisfies the following conditions. According to the optical experiment by the present applicant, imaging is performed with an optical system in which the square of (NA ÷ β) expressed by the numerical aperture (NA) of the objective lens and the projection magnification (β) is 0.01 or more. Thus, it was proved that an image can be formed only by luminescence generated from a single cell (see Japanese Patent Application No. 2005-267531). Furthermore, the present applicant has proceeded with studies and discovered that the variation pattern of gene expression is different among a plurality of cells cultured in the same petri dish (see Japanese Patent Application No. 2005-44737). Further, according to the optical condition investigated by the inventor, when the optical condition expressed by the square of the numerical aperture (NA) / projection magnification (β) of the objective lens of the imaging apparatus is 0.071 or more. It was found that a cell image that can be imaged within 1 to 5 minutes and can be analyzed is provided. A light emission analysis system that observes these light emission images with a storage-type imaging device under a microscope is called a light emission microscope. The light emission microscope preferably has a light shielding means having an opening / closing lid (or an opening / closing window) for shielding light, and a necessary biological sample is set or exchanged by opening / closing the light shielding means. Depending on the purpose, an operation for chemically or physically stimulating a vessel containing a biological sample may be performed manually or automatically. In the best mode, the luminescence microscope is equipped with a known or unique culture device. The culture apparatus has a function of optimally maintaining the temperature, humidity, pH, outside air component, medium component, and culture solution component so as to enable analysis in the system for a long period of time.

  Therefore, the analysis method of the present invention is a method for analyzing intracellular / extracellular interactions based on image information, wherein at least one of the interactable substances is labeled with a luminescent substance as a biological excitation substance. Including the step of causing the agent to be present in one or more cells, optically imaging the resulting emission of the interaction, and imaging the captured optical information for a single cell, wherein the image representation is an interaction It is characterized by the presence and / or amount of weak light caused by it.

In the present invention, the following application ranges are included.
(1) Assay Items In the following wide range of assay items, low invasiveness and direct analysis using weak luminescence can be performed in detail and accurately. For example, gene expression abnormalities (eg determination of gene expression frequency and / or monitoring of fluctuations), internal rhythm disorder related diseases (eg sleep disorders, overwork syndrome, jet lag), temporal pharmacological responses (eg drug sensitivity or drug metabolic activity) Diurnal fluctuations), sunshine response rhythms (eg plant growth rate, phototactic motor activity), chemical response evaluation (drug discovery screening, anticancer drug effect monitoring, post-surgical follow-up of cells (or biological tissues) after transplantation or gene therapy , Water (or serum) stress evaluation), physical stimulus response evaluation (heat shock, electric shock, pressure shock), and developmental biological activity evaluation.

(2) Assay principle Nuclear translocation, receptor receptor signal transduction, nerve cell growth, and biological circadian rhythm.
Among these, in the case of using an optical or thermal stimulus in the assay, the conventional fluorescence detection causes an extra light stimulus or a heat increase due to multiple excitation light irradiations, but because it is a cold light in the luminescence, There is almost no influence on the cell by excessive exogenous beam and heat rise.

(3) Usable devices An imaging device (for example, an optical microscope, a photomultiplier type imager (photomal), an image cytometer), and a spectroscopic measurement device (for example, a luminometer, an integrating sphere photometer, a photon counter) can be mentioned. Among these, the image pickup apparatus is preferably a CCD camera (excellent cooling performance when sensitivity reduction due to incubator temperature is a problem in a system integrated with a culture apparatus) rather than image generation by photomultiplier. ) Is preferable in that a clear image quality can be easily obtained in a short time. However, in measuring biological activity, data can be obtained by any of the imaging apparatus and the spectroscopic measurement apparatus. For example, a light emission amount (or light emission intensity) sufficient to show biological activity can be accumulated before an image is generated in the imaging device. Therefore, when a luminescent image is acquired using any one of the imaging means, it is possible to obtain highly sensitive luminescent data using the same luminescent image. When performing continuous or time-lapse (or time-series) analysis, image generation is performed at least once (preferably the first time) of the emission signals among a plurality of different times, otherwise the image is generated. If only light emission data can be repeated preferably for a designated part or partial region without generation, it is possible to further improve the efficiency of evaluation or analysis time. In the present invention, when a spectroscopic measurement device is used, a specific single cell can be obtained by combining with a means (for example, an optical aperture) capable of measurement limited to a specific part or partial region of a biological sample. The received light data that is smaller than the maximum light-receiving area and contains as little extra area as possible can be obtained continuously or over time by a slightly larger small frame or a large frame including a cell population having a certain extent. Photometry may be repeated repeatedly (preferably in time series).

(4) Target of assay (sample)
Examples include cells or tissues derived from eukaryotes and cyanobacteria. In medical use, a sample containing cells excised by biopsy from a site to be examined in mammals, especially humans is particularly exemplified. In regenerative medicine, at least a part of an artificially modified or synthesized biological sample can be used for the purpose of examining whether biological activity is maintained well. In another aspect, the assay subject of the present invention is not limited to cells or biological tissues derived from animals, but may be cells or biological tissues derived from plants or insects. In the case of bacteria and viruses, the analysis of each part in the container that has not been performed by a conventional luminometer can be targeted. In the luminometer, a large amount of light emission is obtained by stacking an infinite number of samples (for example, 1 million or more per well) in a container such as a well or a petri dish. In the present invention, individual cells or living tissues can be analyzed by storing them in a container at a density that allows individual cells to be identified. Since individual analysis can include a step of calculating the number of only luminescent cells, an accurate interaction per cell can be evaluated.

(5) Inventive information obtained by the present invention One or more kinds of luminescence data generated from each individual cell. As an example, a method and apparatus for statistically elucidating that each cell exhibits a different light emission amount or light emission distribution is provided. As another example, a method and apparatus for a multi-assay that elucidates whether different types of luminescence from the same cell occur at the same or different times is provided. Regarding the point at which multicolor emission analysis can be performed on an image, it is incorporated in the present invention by citing Japanese Patent Application No. 2005-122768 by the applicant, and detailed description thereof is omitted. JP-A-2005-192565 described above discloses an experimental technique in which two types of photoprotein genes of different origins are simultaneously subjected to a luminescent reaction in a well containing a large number of cells (6 million per well). You may refer.

  As another example, a method for elucidating a plurality of identical or different cell populations classified by the amount of luminescence in the same imaging region, and performing a graphic expression such as a histogram or a normal distribution on the classification results as necessary, and Providing equipment. As another example, there is provided a method and apparatus for stably and accurately analyzing various changes in a specific region of a same cell (or tissue) or a region having a certain spread. Provided is a method and apparatus for elucidating a plurality of identical or different cell populations classified by the amount of luminescence in a distribution state imaging region, and performing a graphic expression such as a histogram or a normal distribution on the classification results as necessary.

    By evaluating the interaction using the luminescence image, it is possible to perform a stable quantitative analysis that was difficult in the fluorescence analysis. Moreover, the light emission analysis can be performed with high accuracy by individually analyzing a plurality of captured cell images in the same visual field. Further, by performing the interaction substance in different parts of the same cell or the same tissue by luminescence analysis, it is possible to evaluate a more detailed interaction of minute parts directly and with low invasion. Also, a plurality of different kinds of interacting substances in the same cell (or living tissue) or a plurality of kinds of specific substances (or distribution states) are analyzed for each interacting substance based on image information

  In the following description, Japanese Patent Application No. 2005-267531 and Japanese Patent Application No. 2005-44737 by the applicant can be referred to for details of optical conditions.

Example 1 Induction of Gene Expression by Tetracycline An example of a reporter assay over time in a single cell is described.
In this reporter assay, first, a vector “pcDNA6 / TR (manufactured by Invitrogen)” that constantly expresses tetracycline repressor (TetR) and an expression vector “pcDNA4 / TO (Invitrogen) having a tetracycline operator (TetO2)” are used. And a luciferase gene-linked plasmid are co-expressed in HeLa cells to prepare a specimen. In this state, as shown in FIG. 1, since the TetR homodimer is bound to the TetO2 region, transcription of the luciferase gene is suppressed. Next, as shown in FIG. 2, tetracycline is added to the culture medium to bind to the TetR homodimer, and by changing the three-dimensional structure of the TetR homodimer, the TetR homodimer is separated from TetO2 to induce transcription of the luciferase gene. To do. The culture solution is a D-MEM medium containing 10 mM HEPES and contains 1 mM luciferin.
The lens used as the objective lens and the imaging lens is a commercially available microscope objective lens having specifications of “Oil, 20 ×, NA 0.8” and “5 ×, NA 0.13”, and corresponds to the magnification Mg. The overall magnification is 4 times. The camera used as the camera C1 is a digital camera for microscopes “DP30BW (manufactured by Olympus Corporation)” cooled at 5 ° C., and the CCD element as the CCD 3 is 2/3 inch type, the number of pixels is 1360 × 1024, and the pixel size is 6 μm. It is a horn.

    FIG. 3 is an image obtained by exposing an image of self-emission of a specimen 9 hours after addition of tetracycline for 1 minute. Observation images after 0, 2, 4, 6, 8, and 10 hours have elapsed after the addition of tetracycline. In addition, since these specimens are mounted in an incubator, these observations are performed in an environment of 37 ° C.

  FIG. 4 shows a state in which the four partial areas ROI-1, ROI-2, ROI-3, and ROI-4 designated on the image are sequentially displayed in enlarged frames. FIG. 5 is a diagram showing the results of measuring the change over time in the luminescence intensity of the luciferase gene for each of these four regions. As shown in FIG. 5, luminescence was captured 2 hours after the addition of tetracycline, indicating that a plateau was reached after 6-7 hours. As described above, according to the weak light sample imaging unit and the weak light sample imaging device according to this embodiment, the position of the luciferase gene that emits light is identified and tracked in time series, and the temporal change of the luminescence phenomenon is measured. Can do.

Example 2 HSP70B gene promoter assay
HSP70B (Heat shock Protein70B) is a stress protein whose expression is induced by environmental factors such as heat, toxic substances, and oxygen deficiency. HSP not only contributes to the regulation of cell functions as a molecular chaperone, but also transmits intracellular information. It is also attracting attention as a substance. Therefore, in this example, a vector in which the HSP70B promoter sequence was introduced into pGL3-Basic Vector (Promega) was used for the reporter assay (FIG. 6). When stress such as heat shock is applied to the cells into which this vector has been introduced, the heat shock factor binds to the HSP70B promoter portion and transcription is initiated (FIG. 7). HeLa cells cultured in a glass bottom dish were transfected with the vector by the lipofection method. After overnight culture, 1 mM Luciferin was added, and after heat stimulation (43 ° C., 60 minutes), time-lapse measurement was performed every 30 minutes in a luminescence microscope for 20 hours. As the medium, D-MEM5% FCS-10 mM HEPES was used. First, after obtaining the cell shape and position information by bright field observation, a luminescence image was obtained. The objective lens was a 20x lens, and the exposure time was 5 minutes.
A part of the superimposed image of the light emission image and the bright field image is shown in FIG. Luminescent cells are shown in yellow pseudocolor.

Five regions (ROI) where one cell enters were set (FIG. 9), and the data were converted into numerical values and average values were plotted (FIG. 10). The promoter activity was highest at 10-15 hours after heat shock, but there was a considerable difference in the rate of increase in promoter activity and the time required for activity increase among 5 cells. FIG. 10A shows a cell group in which the promoter activity becomes maximum around 14 hours after stimulation, and FIG. 10B shows a cell group in which the promoter activity becomes maximum around 10 hours after stimulation. In the observation with a conventional luminometer, since a certain number of cells are analyzed together, transfection efficiency, differences between cells, and the like are averaged. However, as shown in FIG. 8, there are actually many untransfected cells, and there are also differences in expression depending on the cells. If data for each cell can be measured with a luminescence microscope, the analysis can be performed in consideration of the difference in transfection efficiency and the influence of the cell cycle.

Example 3 Observation of Subcellular Localization Results of observation of intracellular localization in organelles are shown in FIG. HeLa cells were transfected with a pGL3 vector into which a signal sequence for localization to the nucleus, endoplasmic reticulum, and cell membrane was introduced. After overnight culture, 1 mM Luciferin was added to D-MEM5% FCS-10 mM HEPES medium and a light emission microscope. Was observed. A bright field image, a luminescent image, and an image obtained by superimposing the bright field and the luminescent image are shown. Localization to the target location was observed in each of the nucleus, endoplasmic reticulum, and cell membrane. If the intracellular localization can be detected, for example, it is possible to observe the transition from the cytoplasm to the nucleus when a certain stimulus is given to the cell (FIG. 12). For example, detection of nuclear translocation or the like when a glucocorticoid receptor, which is an intranuclear receptor, is expressed and treated with a glucocorticoid hormone.

Example 4 Analysis of interaction between different cells In the conventional luminescence detection, the amount of luminescence averaged by a luminometer was measured. Interaction analysis is also possible. An example is analysis of nerve cells and glial cells. The nervous system is mainly composed of two types of cells: neurons and glial cells, which play a central role in brain information processing through electrical activity and synaptic connections. Glial cells do not have such electrical activity and are thought to play a role in supporting nerve cell activity by surrounding nerve cells. Many of the roles of glial cells around nerve cells have not been clarified yet.

Example 5 Luminescent Reporter Assay in Tissues In addition to luminescent reporter assays in cells, subjects include tissue samples. For example, by using a brain slice tissue, it is possible to capture different periods and phases for each cell in the suprachiasmatic nucleus, which is the place of a biological clock and brings a difference between day and night to the living thing.

  The present invention is not limited to the embodiments described above, and the following changes or improvements are possible. For example, although a photoprotein that functions as a gene is used as a luminescent substance, another bioluminescent substance having no gene function may be used. The present invention preferably includes a step of imaging while accumulating with a storage-type imaging device a photomarker substance that is minimal for the biological activity of a biological sample (for example, a cell or tissue). This imaging process obtains sample images with weak light that cannot be imaged with conventional scanning (scanning) high-intensity analysis such as fluorescence analysis. Emission analysis can be performed without giving Here, for a purpose different from light emission, a fluorescent substance or chemiluminescent substance having an extremely low concentration that is comparable to or less than half of the light emission may be used in combination. Further, the total amount per single sample may be distributed between the luminescent material and the fluorescent material at a desired ratio, and the total amount of luminescence may be adjusted to be the minimum necessary for storage-type imaging.

 Examples of bioluminescence (or chemiluminescence) that can be used in the present invention include cells or tissues into which DNA containing a luciferase gene has been introduced as a reporter gene linked downstream of a promoter of a specific gene region of interest. . By using a cell or tissue in which luciferase is expressed as a reporter, it is possible to detect changes in transcription over time by detecting luciferase activity at a desired expression site.

  A preferred embodiment is a vertebrate cell or tissue in which the introduced luminescent gene is expressed in a rhythm in peripheral tissues. Peripheral tissues include, but are not limited to, liver, lung, and skeletal muscle. These peripheral tissues are reported to have a circadian rhythm with a phase difference of 7-12 hours. The delay pattern of circadian rhythm is thought to reflect the normal coordination of the biological rhythm of complex mammals composed of multiple organs.

  According to this, the information analyzed according to the present invention is intended to elucidate the mechanism of jet lag or sleep disorder related to circadian rhythm, and to screen and test compounds useful for the treatment of circadian rhythm disorder. It can be said that it is useful for developing a mammal model to be used as

  Moreover, various tests or screenings can be performed by using a transformant or a transgenic mammal containing the DNA of the present invention that expresses a reporter gene. By detecting reporter gene expression in these tissues or cells under a variety of arbitrary conditions, it is possible to assess or screen for the effects of stimuli or compounds that modulate reporter gene expression . Stimulation includes temperature, light, exercise, and other shocks. There are no restrictions on the compounds used. In particular, the present invention relates to a compound that modifies the expression induced by the promoter of a clock gene (for example, Period 1) introduced into the transformant or transgenic mammal of the present invention, and the transformant or transgenic mammal. It is applicable to the method of using or testing.

Examples of the test or screening method of the present invention include the following methods.
Method (1): A method for testing or screening a compound having an activity of altering the expression of a transgene in the transformant of the present invention, comprising: (a) treating the transformant with the compound; b) measuring the expression of the transgene in the treated transformant.
Method (2): A method for testing or screening a compound having an activity of altering transgene expression in a mammal of the present invention, comprising: (a) treating the mammal with the compound; and (b) Measuring the expression of the transgene in the treated mammal.

The method of the present invention is useful for screening compounds that modulate the expression of Period 1 gene. The method is also useful for screening pharmaceuticals for circadian rhythm disorders. Therefore, in addition to the above methods (1) and (2), the following screening methods are also possible according to the present invention.
Method (3): A method for testing or screening a pharmaceutical agent useful for the treatment of circadian rhythm sleep disorder, wherein (a) treating the transformant or transgenic non-human mammal of the present invention with the pharmaceutical agent; And (b) measuring the expression of a reporter gene in the treated transformant or mammal.

  There is no particular limitation on the compound used in the test or screening method of the present invention. Examples include inorganic compounds, organic compounds, peptides, proteins, natural or synthetic low molecular compounds, natural or synthetic high molecular compounds, tissue or cell extracts, microbial culture supernatants, plants or marine organisms. Natural ingredients derived from, etc. are included, but are not limited thereto. Expression products such as gene libraries or cDNA expression libraries may be used. There is no particular limitation on the method of treatment with the compound. In vitro treatment may be performed, for example, by adding the compound to the culture medium and contacting the cell with the compound, or introducing the compound into the cell using a microinjection or transfection reagent. Methods of treatment in vivo include intraarterial injection, intravenous injection, subcutaneous injection, or intraperitoneal injection; oral administration, enteral administration, intramuscular administration, or intranasal administration; ocular administration; injection or catheterization. Methods known to those skilled in the art, such as intracerebral administration, intraventricular administration, or peripheral organ administration, are included. The compound is suitably administered as a composition. For example, it can be mixed with water, saline, buffers, salts, stabilizers, preservatives, suspending agents and the like.

  Reporter gene expression can be measured while the mammal or cell is alive, or can be measured after solubilizing the cell. For example, to measure the expression of a luciferase gene in live tissue, Yamazaki, S. et al. (Science, 2000, 288) incorporated by reference as described in the Examples or by reference herein. 682-685), it is possible to measure bioluminescence continuously. The luciferase activity in solubilized tissue or cells can be measured using, for example, a Luciferase Reporter Assay System (Promega). Reporter gene expression can be measured temporally or spatially. Expression can also be analyzed by detecting the phase, amplitude, and / or period of the expression rhythm. The method of the present invention makes it possible to evaluate the immediate or long-term effects (including phase changes) of a compound. If these expressions are altered by administration of a compound, the compound becomes a drug candidate that regulates the expression of the Period 1 gene. Such compounds are expected to be applied as pharmaceuticals against various circadian rhythm disorders including sleep disorders. For example, an agent that resets or initiates a reporter gene expression oscillation would be expected to reverse or advance the pacemaker phase. Thus, these agents can be used to direct a desynchronized expression pattern to normal synchronization. The pharmaceutical product screened by the present invention is administered to the transgenic mammal of the present invention induced to become a circadian rhythm disorder model in order to evaluate the therapeutic effect of the drug.

  When detecting gene expression in a transgenic mammal, the organ to be measured is not particularly limited, and this includes the central nervous system (CNS) and the peripheral nervous system (PNS) including the hypothalamic SCN, liver, lung, And other peripheral tissues including but not limited to skeletal muscle. The system disclosed in the present invention is useful for assessing the phase relationship and synchronization mechanism of Period 1 expression in SCN and peripheral tissues.

  The system of the present invention can be used to identify a number of factors presumed to modulate Period 1 expression. Once new in vivo factors and genes related to circadian rhythm are identified using the system of the present invention, the in vivo oscillations of expression of these factors and genes can be assessed. Thereby, it is possible to isolate factors that control the vibration phase of SCN and peripheral tissues. These are considered to be novel genes and proteins involved in circadian rhythm, and by using these as targets, it is considered that screening for new drugs becomes possible. Such screening can be done both in vivo and in vitro.

Specifically, the in vivo screening method using the transgenic mammal of the present invention is achieved by the method (4) including the following steps.
Method (4): (a) administering a compound to a transgenic mammal whose circadian rhythm has already been determined; (b) periodically detecting the expression level of the reporter gene in the transgenic mammal, and expressing the rhythm (C) comparing the expression rhythm of the reporter gene after administration of the compound with that before administration; and (d) selecting a compound that alters the phase, period, or amplitude of the expression rhythm.

  The expression rhythm of the reporter gene can be determined by a method of detecting the expression rhythm of the reporter gene in the living animal body; a method of continuously observing expression fluctuations by culturing the excised tissue; or an extract of animal tissue It can be detected by a method that is prepared periodically and detects the expression level at each time point. For example, luciferin is administered to a transgenic animal at an appropriate timing by a suitable method (eg, intravenous injection, intraperitoneal administration, intraventricular administration, etc.). Subsequently, the animal is anesthetized, and the luciferase luminescence is measured by a CCD camera to determine the expression site and expression level of the reporter gene. This measurement is performed several times every few hours to confirm the expression rhythm of individual animals (Sweeney TJ et al., “Visualizing the kinetics of tumor-cell clearance in living animals ) ", PNAS, 1999, 96, 12044-12049; and Contag PR et al.," Bioluminescent indicators in living mammals ", Nature Medicine, 1998, 4, 245-247. ).

As described above, a novel drug can be screened in vitro using the present invention. Such an in vitro screening method can be achieved by the method (5) including the following steps.
Method (5): (a) culturing a tissue or cell derived from the transformant of the present invention or the transgenic mammal of the present invention; (b) compound of the transformant or tissue or cell over an appropriate period of time; (C) a step of periodically detecting the expression level of the reporter gene; and (d) the expression rhythm (phase, cycle, and amplitude) of the reporter gene after the treatment of (b). Selecting a compound to be modified.

  As used herein, the tissue or cell to be imaged of the present invention may be a primary culture or an established cell line cell. Although there is no restriction | limiting in the tissue, cell, etc. which are used by this specification, SCN in a vertebrate, a hypothalamic marginal cell, a peripheral nerve, etc. are preferable. The treatment with the compound may be performed, for example, by immersing tissues, cells, and the like for a certain period in a solvent to which the compound has been added. When measuring the change in the expression rhythm of the reporter gene, comparison may be performed using the same tissue or cell in which the expression rhythm has been determined in advance, or a control tissue or cell that has not been treated with the compound.

  In the in vivo and in vitro screening methods described above, stimulation treatment such as light stimulation may be performed together with administration or treatment of the compound.

  A compound identified by the test or screening method of the present invention can be used as a pharmaceutical agent for a desired circadian rhythm disease or disorder. These agents can be formulated as pharmaceutical compositions by appropriately combining with pharmaceutically acceptable carriers, solutes and solvents. The drug can be applied to diseases or disorders such as jet lag syndrome, sleep disorder due to shift work, sleep phase regression syndrome, and irregular sleep-wake disorder.

  When the compound isolated by the screening method of the present invention is used as a pharmaceutical, it can be directly administered to a patient, or it can be formulated into a pharmaceutical composition by a known pharmaceutical formulation method. You can also. For example, it can be administered after appropriately combining it with a pharmaceutically acceptable carrier or vehicle, specifically, sterilized water, physiological saline, vegetable oil, emulsifier, suspending agent and the like. The pharmaceutical composition of the present invention may be in the form of an aqueous solution, tablet, capsule, troche, buccal tablet, elixir, suspension, syrup, nasal solution, inhalation solution and the like. The content of the compound may be determined as appropriate. These can be administered to a patient, for example, usually by intraarterial injection, intravenous injection, subcutaneous injection, or oral administration, and such methods are known to those skilled in the art. The dose varies depending on the patient's weight, age, administration method, and symptoms, but those skilled in the art can appropriately select the dose. In general, the dosage varies depending on the effective blood concentration of the drug and the metabolic time, but the daily maintenance dose is about 0.001 mg / kg to 1 g / kg, preferably 0.01 mg / kg to 100 mg / kg, more preferably Is considered to be 0.1 mg / kg to 10 mg / kg. Administration may be from once to multiple times per day. If the compound can be encoded by DNA, the DNA can be incorporated into a gene therapy vector for gene therapy.

1 is a schematic diagram of a plasmid in which a luciferase gene is linked to an expression vector having a tetracycline operator (TetO2) in Example 1. FIG. FIG. 3 is a schematic diagram showing a state where TetR homodimer is separated from TetO2 by the action of tetracycline. It is the light emission image for every elapsed time obtained after adding tetracycline with respect to the HeLa cell which introduce | transduced the luciferase gene of Example 1. FIG. In Example 1, it is a figure which shows the state which designated 4 area | regions which should be measured with respect to the whole visual field of the light emission image for observation obtained with a 20 times objective lens. It is a figure which shows the result of having measured the time-dependent change of the luminescence intensity of the luciferase gene about the area | region ROI-1, ROI-2, ROI-3, ROI-4 shown in FIG. It is the schematic diagram which connected the HSP70B sequence which is a heat shock protein to the luciferase gene sequence through the TATA box. It is a schematic diagram which shows the state by which transcription | transfer is started from the HSP70B promoter part by the effect | action of a heat shock factor. In Example 2, it is a figure which shows the image which overlap | superposed the illumination image and light emission image of the cell which passed 60 after heat stimulation (The transfected cell is light-emitted and is expressed in white in the figure, but most other than that is the figure. Cells are not transfected). In Example 2, five regions (regions surrounded by solid lines) to be measured with respect to the entire visual field of the observation light emission image obtained by the 20 × objective lens and the superimposed image regions (dotted lines) shown in FIG. It is a figure which shows the state which designated the area | region enclosed by (). It is the figure which expressed the time-dependent data of the light emission amount based on the light emission image which carried out the type-lapse imaging for every 5 area | regions designated in Example 2 graphically. For individual cells in each region, the promoter activity due to heat shock becomes maximum at around 14 hours in the cell group (ROI1, ROI2) (FIG. 10A), and maximum at around 10 hours in the cell group (ROI3, ROI4, ROI5). (FIG. 10B). It is a figure (the bright field image, the luminescent image, and the superposition image of bright field and luminescence in order from the left) which shows the luminescence image localized in the cell for every organelle (nucleus, endoplasmic reticulum, cell membrane). It is a figure which shows a mode that it transfers by the stimulation process which induces a nuclear transfer in the cell which expressed the nuclear receptor.

Claims (17)

In the method of analyzing an interaction between cells and tissues, an interaction substance in which a luminescent substance as a biological excitation substance is labeled in at least one of the substances that can interact is present in a plurality of cells, and is opened. When the number and the projection magnification are expressed as NA and β, respectively , using an objective lens with a value of (NA / β) ² of 0.01 or more, the light emission resulting from the interaction includes imaging of a plurality of cells A method for analyzing biological interaction, comprising the steps of: imaging while optically imaging with a field of view, and individually analyzing a plurality of cell images in the same field of view.   The method of claim 1, wherein the presence and / or amount of faint light in the individual cell image correlates with an interaction.   2. The analysis method according to claim 1, wherein the biological excitation substance includes a component that causes bioluminescence.   4. The analysis method according to claim 3, wherein the component that causes bioluminescence uniformly contains a substrate for a luminescence reaction.   3. The method according to claim 1 or 2, further comprising the step of evaluating an interaction with a chemical stimulant that affects the activity of the biologically excited substance.   3. The method according to claim 1, further comprising the step of evaluating an interaction with a physical stimulus that affects the activity of the biologically excited substance.   The method according to claim 1, wherein the biological sample contains living cells.   The method according to claim 1, wherein the analyzing step includes a measuring step of statistically measuring a plurality of cells in the same visual field.   9. The analysis method according to claim 8, wherein the measuring step includes a step of calculating the number of cells producing at least detectable luminescence.   The method according to claim 9, wherein the counting step includes a step of sorting by group according to the amount of luminescence for each cell.   11. The analysis method according to claim 8, further comprising a step of expressing the result information obtained by the measurement step in association with a cell image so as to be visible.   11. The method according to claim 8, wherein the imaging process is executed at a number of different timings, and result information from the measurement process is output in real time along a time axis, or an activity of a luminescence reaction is performed. An analysis method including a step of outputting in synchronization with a curve. In the method for analyzing the interaction between cells and tissues, at least one of the interactable substances, the interacting substance labeled with a luminescent substance as a biological excitation substance is used as a plurality of different sites in the same cell or the same tissue. As a result of the interaction , an objective lens having a value of (NA / β) ² of 0.01 or more when the numerical aperture and the projection magnification are expressed as NA and β respectively. A step of analyzing the information on the same cell or a plurality of parts in the same tissue based on the imaged image information. A method for analyzing a characteristic biological interaction. In a method for analyzing an interaction between cells and tissues, at least one of the interactable substances, an interaction substance labeled with a luminescent substance as a biological excitation substance is placed in a specific place of the same cell or the same tissue. Or a light emission as a result of an interaction using an objective lens having a value of (NA / β) ² of 0.01 or more when the numerical aperture and the projection magnification are expressed as NA and β, respectively. Of the light-emitting substance based on image information obtained by imaging information on a plurality of parts in the same cell or the same tissue imaged in time series while optically imaging the imaging field including the cells or tissues. A biological interaction analysis method comprising a step of analyzing a change in a place or distribution state. In the method of analyzing interactions inside and outside a cell or tissue, a plurality of types of interacting substances capable of interacting are allowed to exist in the cell or tissue in a state where a luminescent substance as a biological excitation substance is labeled, and the numerical aperture and When the projection magnification is expressed as NA and β, respectively , using an objective lens having a value of (NA / β) ² of 0.01 or more, the luminescence resulting from the interaction is included in the imaging field including the cells or tissues A method for analyzing biological interaction, comprising the steps of: taking an image while optically forming an image by using the image information, and analyzing each of the interacting substances based on the taken image information.   16. The analysis method according to claim 15, wherein the plurality of types of interacting substances are separately labeled with luminescent substances having different optical characteristics that are optically distinguishable.   16. The analysis method according to claim 15, wherein chemical or physical stimuli specific to each of the plurality of types of interacting substances are executed at different timings.